Highly porous magnetic temporary fixed bed

ABSTRACT

The present invention relates to a fixed bed, for example for the isolation and/or purification of components originating from a biological system, which fixed bed comprises magnetic beads and a magnetizable fabric arranged at least in part in the fixed bed, a fluidized bed-fixed bed, which comprises the fixed bed of the invention after application of an alternating magnetic field, and also to a process for the isolation and/or purification of components originating from a biological system.

The present invention relates to a fixed bed, for example for theisolation and/or purification of components originating from abiological system, which fixed bed comprises magnetic beads and amagnetizable fabric arranged at least in part in the fixed bed, afluidized bed-fixed bed (also “temporary fixed bed/fluidized bed”),which comprises the fixed bed of the invention after application of analternating magnetic field, and also to a process for the isolationand/or purification of components originating from a biological system.

An essential aspect of pharmaceutical biotechnology is the isolation orpurification of components, for example proteins, originating frombiological systems. This isolation or purification customarily proceedsvia chromatographic or fixed-bed adsorption processes in which thecomponents to be isolated or purified are bound to beads functionalizedin a suitable manner. The sorption rate and thus the efficiency of theprocess depends, inter alia, on the bead size used and also on the flowvelocity of the mobile phase through the fixed bed.

Although the beads customarily used in the fixed-bed adsorption process,at 35 to 50 μm, have a suitable size for rapid adsorption of thecomponents originating from biological systems, the capacity of suchsystems is greatly restricted, however, owing to the comparatively lowflow velocities of the mobile phase through the fixed bed. In addition,fixed-bed adsorption processes require a usually complex pretreatment(filtration, centrifugation or sedimentation) of the suspensioncontaining the product in order to obtain a solids-free solution, sinceotherwise the fixed bed becomes blocked.

The complex pretreatment of the suspension containing the product can,however, be avoided by the use of fluidized-bed-based adsorbers(expanded bed adsorption, EBA), since fluidized beds, in contrast tofixed beds, cannot be blocked by solid particles, such as cellfragments, for example, present in the suspension. However, thedevelopment/generation of fluidized beds is only possible usingcomparatively large beads in the range from 150 to 250 μm. Owing to thelow sorption rates of biomolecules such as proteins, for example,therefore only superficial loading of the beads proceeds, which leads toa low efficiency of this process. A further disadvantage of fluidizedbeds is the axial mixing of the fluid and of the beads. In addition,inhomogeneous particle-free regions can lead to impairments inconversion rate.

By using magnetic beads in magnetically stabilized fluidized beds(MSFBs), the fluidized-bed processes can be significantly improved.Magnetic beads are microparticles which have a magnetic core and afunctionalized surface.

In the MSFB, magnetic beads are fluidized in a column by an upwardlydirected liquid stream. In this case the magnetic beads are heldstationary within the fluidized bed in magnetic bead chains by amagnetic field. With strong magnetic forces, a magnetic fixed bed forms.As a result of the magnetic stabilization, it is possible to essentiallysuppress the axial mixing caused by the bead movement. As a result,higher flow velocities compared with customary fluidized beds arepossible, which leads to higher efficiency of the system.

By means of the MSFB, low axial mixing, low pressure drop, good masstransfer behaviour, high throughputs and continuous operation may beachieved. However, in the MSFB there is a tendency towards channelformation, as a result of which the efficiency of the system isdecreased. In addition, owing to the fixed-bed-like character at highmagnetic field strengths, the problem of blocking of the beads by solidspresent in the mobile phase can also occur.

In addition to the MSFB, fluidized beds of magnetic beads in analternating magnetic field are also known (magnetically stirred reactor,MSR). In this case, a strong vortexing of the magnetic beads is achievedby means of the alternating magnetic field. This results in a fluidizedbed which can be maintained even without a fluid stream.

Therefore, the object of the present invention is to provide a novelsystem for the isolation and/or purification of components originatingfrom a biological system using magnetic beads (also “magneticparticles”), which system combines the advantages of a fluidized bedsystem with those of a fixed bed system, and which in particular makespossible low axial mixing, low pressure drop, good mass-transferbehaviour, high throughputs and continuous operation without apretreatment of the biological system by filtration or the like.

This object is achieved by the embodiments characterized in the claims.

In particular, a fixed bed, for example for the isolation and/orpurification of components originating from a biological system isprovided, comprising magnetic beads having a particle size in the rangefrom about 10 to about 100 μm which is capable of the binding oradsorption of components originating for example from a biologicalsystem and a magnetizable fabric arranged at least in part in the fixedbed having a mesh width which corresponds to the particle size range ofthe magnetic beads respectively present in the fixed bed.

In this case the system can be a suitable biological system. Forexample, the biological system is a digest (also “lysate”) originatingfrom prokaryotic or eukaryotic cells, a cell culture supernatant, a cellculture, a biological liquid or suspension, or a protein mixture ornucleic acid mixture.

The components originating from the biological system which are intendedto be isolated and/or purified can be all components which are ofinterest biotechnologically and can bind magnetic beads modified in asuitable manner. For example, the components are biomolecules such asenzymes, proteins, (poly)peptides, lipids, sugar-containing compounds ornucleic acids. These biomolecules can also have a labelling which, interalia, indicates higher selectivity and/or affinity to the magnetic beadshaving a corresponding surface modification. Examples of these are whatare termed fusion proteins having “His tags”.

As magnetic beads, use can be made of magnetic beads known in the priorart which are capable of the binding of the components originating fromthe biological system. For this the magnetic beads are surface-treatedin a suitable manner, that is to say the surface of the magnetic beadsis provided with groups having an affinity for the components to beisolated or purified. Such groups are known in the prior art. Inaddition, it is known in the prior art in what manner magnetic beadsmust be surface-treated in order to bind selectively a certain componentfrom a biological system.

The magnetic beads used in the context of the present invention have aparticle size in the range from about 10 to about 100 μm, preferably inthe range from about 10 to about 50 μm. This particle size enables thebinding or adsorption of the components originating from the biologicalsystem to be achieved at a high rate, which significantly improves theeffectiveness of the isolation or purification system.

The magnetizable fabric serves for magnetic fixing of the magneticbeads. Preferably, the magnetizable fabric is a metallic (also“metal-containing”) wire fabric. The magnetizable fabric can passthrough the fixed bed and/or be arranged as outer limit of the fixedbed. In order that the magnetizable fabric effectively fixes themagnetic particles, it is advantageous that the mesh width of the fabricsubstantially corresponds to the particle size of the magnetic beads.The magnetizable fabric can be arranged in this case at any desiredangle to the direction of flow, preferably perpendicularly.

The fixed bed of the invention is preferably arranged in a suitablecontainer or housing. Such housings are known in the prior art and arepreferably available in the form of a column. The housing consists, forexample, of a suitable material such as glass or plastic.

Application of an alternating magnetic field having a frequency in therange from about 0.01 to about 20 Hz, preferably at a frequency in therange from about 0.05 to about 10 Hz, produces a fluidized bed-fixed bedfrom the fixed bed of the invention. In the context of the presentinvention, a fluidized bed-fixed bed (also “temporary fixedbed/fluidized bed”) is taken to mean a system in which the beneficialproperties of a fluidized bed are combined with those of a fixed bed ina synergistic manner.

As a result of the alternating magnetic field, the magnetic beads areregularly rearranged, as a result of which a regular restructuring ofthe bed is achieved. This leads to a bed having fluidized bed character,which has a high porosity and a low pressure drop, in which blocking bysolids such as cell fragments, for example, can be avoided and whichpermits high flow velocities.

Secondly, as a result of the magnetic fixing of the magnetic beads,according to the invention use can be made of beads having a size fromabout 10 to about 100 μm in the bed. As a result, the bed according tothe invention likewise has fixed bed character which enables a highloading of the magnetic beads and also the realization of a plurality ofseparation stages (also “theoretical plates”). In addition, as a resultof the magnetic fixing, countercurrent flow conditions are significantlysimpler to implement than in a fluidized bed or in a fixed bed.

Using the fluidized bed-fixed bed according to the invention it ispossible to combine the advantages of a fluidized bed with thereaction-kinetics advantages of small beads having a size of about 10 toabout 100 μm. In particular, this combination, in addition to a highconvective mass transport, simultaneously makes possible a high reactionrate owing to the favourable surface/volume ratio of the magnetic beads.In addition, as a result of the magnetic fixing of the beads, the usableflow velocity increases to a multiple of the discharge velocity in aconventional fluidized bed. As a result of the arrangement of the beadsalong the magnetic field lines, in addition good flowability through thebed is achieved. This effect is further increased by the magnetizablefabric in the bed.

In addition, the present invention relates to a process, for example forthe isolation and/or purification of components originating from abiological system, which uses the above-defined fluidized bed-fixed bedfor the adsorption or binding of the components originating from abiological system to the magnetic beads.

In this case preferably a mixture which contains the componentsoriginating from a biological system is fed to the fluidized bed-fixedbed according to the invention. After the binding or adsorption of thecomponents originating from a biological system to the magnetic beads inthe fluidized bed-fixed bed according to the invention, the magneticbeads together with the components bound or adsorbed thereto can beseparated off. In the case of continuous operation in countercurrentflow, suitable processes are known in the prior art for separating offthe magnetic beads from the fluidized bed-fixed bed and use, forexample, the magnetic character of the beads. After separating off themagnetic beads from the fluidized bed-fixed bed, the components bound tothe magnetic beads can be eluted by suitable processes. Elution can alsoproceed within the fluidized bed-fixed bed. As a result, thecorresponding components are obtained in isolated or purified form. Themixture which contains the components originating from a biologicalsystem is preferably fed via a countercurrent flow procedure. In thismanner, more efficient isolation or purification can proceed.

FIG. 1 shows a diagrammatic presentation of the fluidized bed-fixed bedaccording to the invention.

The present invention will be described in more detail hereinafter withreference to an example, without, however, being restricted by this.

EXAMPLE Experimental Set-Up

The experimental plant essentially consisted of the reactor, themagnetic coil system and also a measurement data detection system notdiscussed further here.

FIG. 1 shows a diagrammatic sketch of the structure.

Column:

The reactor consisted of a Plexiglas column (internal diameter 18 mm,length 107 mm). The connection of both feed ports was effected via screwthreads and was simultaneously used for fixing the magnetizable wirefabric having a nominal mesh width of 100 μm. The temperature of thesolutions corresponded to the ambient temperature. In the reactoroutlet, the flow rate was set via a rotameter. The flow rate was checkedover an adequate period using a standard cylinder.

Coil Systems for Generating the Magnetic Field:

The column was placed between a coaxially arranged cylindrical coilsystem of two or four coils (M). To generate a homogeneous magneticfield, the coils were mounted apart by the distance of their radius(simplified HELMHOLTZ arrangement). To generate the alternating magneticfield, an amplifier was coupled to an analogue frequency generator. Inthis case the flux density could be varied between 0 and 7 mT and thefrequency between 0 and 1000 Hz.

Magnetic Beads:

The magnetic beads used consisted of a polyvinyl alcohol-acrylic acidmatrix having enclosed maghemite particles (gamma-Fe₂O₃). The remanencewas 21 A·m²/kg. The bead diameter was about 125 μm and the density 1.23g/ml. The surface of the beads was functionalized by imidodiacetic acid(IDA).

Using the above-described device, “HIS-tagged” eGFP was purified orisolated from an E. coli cell digest.

1. Fixed bed, comprising magnetic beads having a particle size in therange from 10 to 100 μm, and a magnetizable fabric arranged at least inpart in the fixed bed having a mesh width which corresponds to theparticle size range of the magnetic beads respectively present in thefixed bed.
 2. Fixed bed according to claim 1, wherein the magnetic beadshave a particle size in the range from 10 to 50 μm.
 3. Fixed bedaccording to claim 1, wherein the magnetic beads are capable of theadsorption of components originating from a biological system.
 4. Fixedbed according to claim 3, wherein the components originating from thebiological system are biomolecules comprising enzymes, proteins,(poly)peptides, lipids, sugar-containing compounds or nucleic acids. 5.Fixed bed according to claim 3, wherein the biological system is adigest originating from prokaryotic or eukaryotic cells, a cell culturesupernatant, a cell culture, a biological liquid or suspension, or aprotein mixture or nucleic acid mixture.
 6. Fixed bed according to claim1, wherein the magnetizable fabric is a metal-containing wire fabric. 7.Fluidized bed-fixed bed, comprising the fixed bed according to claim 1after application of an alternating magnetic field having a frequency inthe range from 0.01 to 20 Hz.
 8. Fluidized bed-fixed bed according toclaim 7, wherein the alternating magnetic field has a frequency in therange from 0.05 to 10 Hz.
 9. Process for the isolation and/orpurification of components originating from a biological system, theprocess comprising the step of adsorption of the components originatingfrom a biological system to the magnetic beads in the fluidizedbed-fixed bed according to claim 7.